We propose to study the mechanism of electron and proton transfer pathways in mitochondrial NADH-quinone oxidoreductase (often referred to as Complex I). this complex plays a central role in the oxidation of NADH, the reducing product of cellular metabolism, by the respiratory chain. Complex I is the most complicated and least understood energy transducing proton-motive enzyme of the respiratory chain. the proposed research takes new directions in the investigation of the mechanism, kinetics, and regulatory properties of this complex in both the isolated and membrane-bound states. The specific studies will include: [I] determination of the minimal structure of Complex I capable of catalyzing NADH oxidation which is coupled with vectorial transfer of protons across the membrane; [II] determination of the internal electron transfer sequence within intrinsic redox components of NADH-quinone oxidoreductase by utilizing artificial electron acceptors (transition metal complexes) with proper redox potentials and appropriate other parameters (hydrophilicity, charge, size); [III] physico-chemical studies on the ubisemiquinones associated with the specific binding sites in Complex I which was recently discovered in Russian Co-P.I.'s laboratory; [IV] studies of molecular events involved in the slow active/inactive transition of Complex I, rediscovered recently by the Russian Co-P.I., which will be extended to searches of physiologically relevant factors which are involved in the control of hysteretic behavior of this complex. The P.I. has extensive experience with EPR studies and with thermodynamic analysis of the intrinsic paramagnetic redox centers of mitochondrial and microbial systems. Specifically, the P.I. has made significant contribution in the characterization of iron-sulfur clusters, flavin and ubiquinone free radicals. The Russian Co-P.I. is an established biochemist and has recently developed unique submitochondrial particle preparations having tightly coupled Site I energy transduction. The collaborative program between these investigators, therefore, is expected to greatly enhance the understanding of the mechanism of energy coupling and its regulation.